Formulation strategies in stabilizing peptides in organic solvents and in dried states

ABSTRACT

The invention relates to stabilized formulations of therapeutically active peptides, particularly PACAP 66. Formulations of the invention include a peptide containing at least one histidine residue, a transition metal salt and an organic solvent. The above formulations may contain peptides that have at least one asparagine residue and are acidified and dried (such as spray-dried or freeze-dried) before formulation preparation. Other formulations of the invention relate to stabilized formulations of PACAP 66 or peptides containing an asparagine residue, which are acidified and dried (such as spray-died or freeze-dried) with or without a transition metal salt.

FIELD OF THE INVENTION

The invention is generally related to the field of pharmaceuticalformulations. More specifically, the invention is directed to stabilizedformulations of therapeutically active peptides in an organic solvent,in an organic solvent-based suspension, or in a dried, such aslyophilized or spray-dried, state.

BACKGROUND OF THE INVENTION

Therapeutic peptides are susceptible to aggregation and/or chemicaldegradation when stored in an aqueous solution for extended periods oftime. This tendency of peptides to aggregate or degrade is generallycharacterized as “instability” and may be measured by many differentanalytical methods, such as UV/VIS spectrophotometry, Reversed PhaseHigh Performance Liquid Chromatography (RP-HPLC), CapillaryElectrophoresis (CE), etc. The instability of peptides in an aqueoussolution may be minimized by a variety of strategies. Wang, Int. J.Pharm., 85:129-88 (1999); Arakawa, et al., Adv. Drug Deliv. Rev.46:307-26 (2001). Two often-used strategies are to formulate thepeptides with a proper amount of a stabilizer(s) or to dry (such asspray-dry, freeze-dry) the peptide for long-term storage.

A rare method of stabilizing peptide for long-term storage is mixing thepeptide with a non-aqueous organic solvent. Organic solvents may improvethe stability of peptides by promoting formation of secondary structures(Zou and Sugimoto, Biometals, 13:349-59 (2000); Kozin, et al., Biochem.Biophys. Res. Commun., 258:959-64 (2001)) and by inhibiting certainchemical reactions, such as hydrolysis (Brennan and Clarke, ProteinSci., 2:331-38 (1993)). Peptide deamidation can be modestly inhibited inan aqueous solution upon addition of an organic solvent, such asglycerol (Li, et al., J. Pept. Res. 56:326-34 (2000)), and ethanol ordioxane (Brennan and Clarke, supra). For example, the stability ofleuprolide, a 9-amino acid peptide hormone, has an overall betterstability in dimethyl sulfoxide (DMSO) than in water. Hall, et al., J.Pept. Res., 53:432-41 (1999); Stevenson, et al., Int. J. Pharm.,191:115-29 (1999).

The native pituitary adenylate cyclase-activating polypeptide (PACAP) isa peptide hormone with less than 40 amino acids. Vaudry, et al.,Pharmacol. Rev., 52:269-324 (2000). Based on its sequence, PACAP is amember of a superfamily of peptide hormones, including vasoactiveintestinal peptide (VIP), glucagon, growth hormone releasing factor(GRF), and secretin (Vaudry, et al., supra). By binding to differentreceptors, PACAP initiates a variety of pharmacological activities, oneof which is the stimulation of insulin secretion. As discussed in arelated application (co-owned, co-pending U.S. Ser. No. 09/671,773, WO01/23420), PACAP without modification is not suitable to treat type IIdiabetes, because significant side effects may occur. In search of aPACAP-like peptide(s) that can be used safely to treat type II diabetes,a variety of PACAP analogues were synthesized and PACAP 66 wasidentified. PACAP 66 is the same molecule as “R3P 66” which is disclosedin U.S. Ser. No. 09/671,773 and in WO 01/23420, both of which areincorporated herein by reference. The peptide sequence for PACAP 66 isHSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY (SEQ ID NO: 1).

The degree of instability of PACAP 66 has, however, been found to be fargreater than what is expected of a peptide in general. In the evaluationof its stability, we found that PACAP 66 was not stable enough in anaqueous environment. Furthermore, addition of a potential formulationstabilizer did not improve its stability. Among the excipients we testedwere different metal ions, such as zinc, magnesium, or calcium, but noneof these ions improved the stability of the peptide. (See FIG. 1.)

In order to overcome this stability barrier and, in turn, increase theproduct shelf life, preparations of the peptide in organic solvents weremade. Fortunately, the peptide dissolved easily in some organicsolvents, but, surprisingly, the stability of this peptide in theseorganic solvents was as poor as, or even worse than, in an aqueousenvironment. (See FIGS. 1 and 2 for comparison.) Many potential peptidestabilizers in an aqueous solution, such as sugars, are not readilysoluble in organic solvents, and therefore could not be used. Many otherknown strategies for peptide stabilization were tried without success.New methods and formulations for the stabilization of PACAP 66 weretherefore needed. Such methods yielded novel formulations and methodsthat are extendible to other peptides. The present invention thereforeprovides novel methods of controlling peptide instability in organicsolvents, in organic solvent-based suspensions, and in dried states.

SUMMARY OF THE INVENTION

The invention provides formulations of peptides either in suspension orsolution, or freeze- or spray-dried, that are stabilized by a transitionmetal salt, an acid or both. In an embodiment of the invention,formulations, either in suspension or solution or dried, include apeptide containing at least one histidine residue and a transition metalsalt. The transition metal salt may be a salt of a transition metalselected from zinc, copper, iron, manganese, nickel or cobalt, and ispreferably zinc. The histidine residue of the peptide may be a terminalhistidine residue. The peptide is preferably PACAP 66, but may includeother peptides, such as, for example, PACAP, PACAP-like peptides, VIP,glucagon, glucagon-like peptides, GRF, secretin, helodermin, exendin-4,and functionally equivalent variants thereof. Also included may beadrenocorticotropic hormone, angiotensins, renin substratetetradecapeptide, natriuretic peptides, gastrointestinal peptides,luteinizing hormone releasing hormone, melanocyte sitmulating hormone,and neurotensin, and parathyroid hormone.

In another embodiment, such formulations of the invention include anorganic solvent. The organic solvent may be; for example, DMSO,1-methyl-2-pyrrolinidone, propanol, propylene glycol, glycerol acetate,monothioglycerol, acetic acid, diethanolamine, benzyl alcohol, ethyllactate, glycerol formal, N-methylpyrrolidone, polyethyleneglycol 400,and isopropyl myristate, or may be a mixture of two or more of thesesolvents. The organic solvent is preferably DMSO,1-methyl-2-pyrrolinidone or propanol. In one embodiment of theinvention, the molar ratio of zinc salt to peptide in the organicsolvent is above 0.1.

In another embodiment of the invention, formulations of the inventioninclude dried formulations containing a peptide having at least oneasparagine residue and an acid. The acid may be TFA or is an inorganicacid, such as, for example, HCl and H₃PO₄. Such formulations may bespray- or freeze-dried. Such formulations may also contain a transitionmetal salt, as described above. In one embodiment of this formulation,the peptide is PACAP 66 and/or a salt thereof Finally, such formulationsmay also contain an organic solvent, as described above.

The invention also relates to processes for manufacturing theformulations detailed above. Such processes include preparing an acidsolution in water, cooling the acid solution to below room temperature,mixing the cooled solution with a peptide containing at least oneasparagine residue, as described above, and then drying the resultingmixture, preferably by spray- or freeze-drying. A transition metal salt,as described above, may be added to the cooled solution before drying.The acids and peptides for use in processes of the invention are asdescribed above.

In another process of the invention, a transition metal salt, asdescribed above, is mixed with a peptide containing at least onehistidine residue, as described above, and then dried, preferably byspray- or freeze-drying. An organic solvent, as described above, mayalso be added to the mixture.

The invention is described in more detail below by the followingdrawings, description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the stability of PACAP 66 in an aqueous solution at 40° C.in the presence of different metal ions.

FIG. 2A shows the stability of PACAP 66 in DMSO at 40° C. in thepresence of different metal ions as analyzed by RP-HPLC.

FIG. 2B shows the stability of PACAP 66 in DMSO at 40° C. in thepresence of different metal ions as analyzed by CE.

FIG. 3 shows the stability of acidified, lyophilized PACAP 66 in DMSO at40° C.

FIG. 4 shows the effect on the stability of PACAP 66 in DMSO at 40° C.of HCl or a combination of HCl and ZnCl₂.

FIG. 5 shows the effect on the stability of PACAP 66 in1-methyl-2-pyrrolinidone at 40° C. of HCl or a combination of HCl andZnCl₂.

FIG. 6 shows the effect on the stability of PACAP 66 in 2-propanol at40° C. of ZnCl₂.

FIG. 7 shows the effect on the stability of lyophilized PACAP 66 at 40°C. of HCl or a combination of HCl and ZnCl₂.

FIG. 8A shows an NMR spectrum of PACAP 66 in DMSO in the absence ofZnCl₂.

FIG. 8B shows an NMR spectrum of PACAP 66 in DMSO in the presence ofZnCl₂.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to stabilized peptide formulations. Peptideformulations of the invention include organic, anhydrous solutions,suspensions, or dried solids, which are stabilized by addition of ametal ion, by acidification and drying of the peptide, or by acombination of the two methods. Specific embodiments of the inventioninclude stabilized formulations of PACAP 66, or “R3P 66” (SEQ ID NO: 1).

PACAP 66 is not stable in an aqueous environment Addition of differentmetals, such as zinc, magnesium, or calcium, does not improve itsstability (FIG. 1). This appears to be caused by peptide autolysis, aswas seen with VIP, a closely related peptide. Mody, et al., Int. J.Pept. Protein Res., 44, 441 447 (1994). In pursuing methods ofstabilizing PACAP 66, we evaluated the stability of this peptide inorganic solvents. We initially found that the stability of this peptidein several organic solvents was unsatisfactory, or even worse than thatobserved in an aqueous environment. (See FIGS. 1 and 2 for acomparison.)

To improve the stability of PACAP 66 in these organic solvents, wedesigned a variety of stabilizing strategies, and a few of these provedto be unexpectedly effective. These include two approaches that turnedout to be very effective in stabilizing the peptide: (1) addition of ametal salt, such as, for example, zinc chloride, in an organic solventand (2) acidification of the peptide in an aqueous solution followed bydrying. The stabilization of PACAP 66 in an organic solvent by zinc saltwas surprising, because several metal salts failed to stabilize PACAP 66in an aqueous solution. (See, e.g., FIG. 1). It was also surprising tofind that the peptide was much more stable in an organic solvent afterthe peptide was acidified and dried, because acidification of a peptidesolution usually leads to increased hydrolysis of the peptide. Thesestabilization strategies were also found to be effective in organicsolvent-based suspensions and in a dried state during storage. In thefollowing section, these successful strategies and the stabilizationmechanisms that made them successful are more fully described. Theimplications of these findings and possible medical uses of theformulations are also described below.

Strategies in Stabilizing Peptides in Organic Solvents

(1) Use of Specific Metal Ions

Different metal salts, including ZnCl₂, MgCl₂, and CaCl₂ were separatelydissolved at 1 mM in DMSO, a non-aqueous organic solvent. PACAP 66 wasthen dissolved in these solutions at 2 mg/mL. The bulk solution wasaliquoted into 2-mL screw-capped (with an o-ring) sterile polypropylenevials. These stability samples were incubated at 40° C. and analyzed atpredetermined intervals.

FIG. 2 shows the stability of PACAP 66 in DMSO as determined by thepeptide recovery (RP-HPLC) and purity (CE) at 40° C. in the presence ofdifferent metal salts. More than 70% of PACAP 66 was degraded in DMSO in4 weeks at 40° C. by RP-HPLC, but only approximately 10% of PACAP 66 wasdegraded in the presence of 1 mM ZnCl₂ under the same storageconditions. The other samples, containing MgCl₂, and CaCl₂ did not haveany significant stabilizing effect when compared with the control. TheCE results were similar to the findings from the RP-HPLC analysis. Thepurity of PACAP 66 in the 4-week control stability sample by CE washigher than the recovery by RP-HPLC, suggesting that certain PACAP 66degradation products might have a different UV response or were not wellseparated from the main peak by CE.

(2) Acidification and Lyophilization of PACAP 66

Several acid solutions were prepared at 0.1%, including HCl, trifluoroacetic acid (TFA), and H₃PO₄ and cooled to 2-8° C. The cold acidsolutions were then mixed with PACAP 66 at a PACAP 66:acid molar ratioof 1:10. After mixing, the cold PACAP 66 solutions were immediatelyplaced inside a precooled freeze-drier and were lyophilized. Thelyophilized material was further equilibrated in a desiccator containingP₂O₅ for at least one day to absorb additional moisture from thelyophilized peptide. The acidified and dried material was then dissolvedin DMSO at 2 mg/mL and stability was conducted as described in the uppersection.

FIG. 3 shows the stability of acidified and lyophilized PACAP 66 inDMSO. More than 50% of unprocessed PACAP 66 was degraded in the controlsample after storage at 40° C. for 2 weeks, while a lower percentage ofdegradation, less than 10%, was observed for samples containingacidified and lyophilized PACAP 66. The relative stabilization effect bythese acids was HCl >TFA >H₃PO₄, in an apparent order of decreasingacidity. The recovery of HCl-acidified PACAP 66 at the end of a 2-weekperiod was 97% by RP-HPLC. However, the recovery could be slightlyoverestimated, as the corresponding purity of PACAP 66 in the sample wasonly 89% by RP-HPLC.

We understand that peptides can be hydrolyzed readily under acidicconditions in an aqueous solution. Secretin, a PACAP-like peptide, canbe degraded easily in an aqueous solution at pH 4. In the acidificationof PACAP 66, the pH of acidified PACAP 66 solution was measured to be2.2 after addition of TFA. At this pH, PACAP 66 should be rapidlyhydrolyzed. However, the acidification process was conducted at a lowtemperature, followed by immediate lyophilization, and no detectablehydrolysis in PACAP 66 was observed.

In the investigation of the degradation mechanisms of PACAP 66 in DMSO,we found that the major degradation pathway of this peptide wasdimerization. The peptide dimer was formed via a cyclic imideintermediate on the asparagine residues in the peptide. Severs, et al.,“Instability of Asparagine and Aspartic Acid of a Polypeptide in DMSO”,WCBP,7th Symposium on the Interface of Regulatory and AnalyticalSciences for Biotechnology Health Products, San Francisco, Calif.(2003). Therefore, acidification of the peptide inhibited dimerizationthrough these amino acid residues in DMSO. (See also Mechanisms of PACAP66 Stabilization, infra).

(3) Stabilization of PACAP 66 at High Concentrations, in Other OrganicSolvents, in Organic Solvent Suspensions, and in Lyophilized State

To test whether a metal salt would stabilize PACAP 66 at a high peptideconcentration in an organic solvent, a high concentration of a metalsalt would be required, assuming a fixed ratio of metal and peptide isneeded for stabilization. A metal salt, however, has limited solubilityin an organic solvent Therefore, a similar preparation method wasadopted for sample preparation of peptide-metal mixtures at highconcentrations, as described under Acidification and Lyophilization ofPACAP 66, supra. Briefly, a metal salt and the peptide were firstdissolved at a fixed molar ratio in an aqueous solution. The solutionwas then aliquoted in 3-mL glass vials at a fixed volume andlyophilized. Stability samples were prepared by adding a fixed amount ofan organic solvent in the vial. The sample vials were then capped,sealed, and incubated at 40° C. Stability samples were first diluted toa reasonable concentration before analysis by RP-HPLC or CE. Similarly,a peptide suspension was prepared by mixing a proper amount of anorganic solvent in a sample vial containing the lyophilized mixture andincubated at 40° C. Stability of solid PACAP 66 was evaluated directlyby incubating the sample vial containing the lyophilized mixture at 40°C.

FIG. 4 shows the stability of PACAP 66 solution at 300 mg/mL in DMSO at40° C. PACAP 66 in the sample was acidified in the absence and presenceof ZnCl₂. Approximately 70% of the peptide was degraded in the controlsample after storage for 23 weeks, while approximately 20% was degradedin the acidified samples in the presence or absence of ZnCl₂.

FIG. 5 shows the stability of PACAP 66 solution at 20 mg/mL in1-methyl-2-pyrrolinidone at 40° C. PACAP 66 in the sample was acidifiedin the absence and presence of ZnCl₂. The peptide was degraded to anon-detectable level in the control sample after storage for 9 weeks,while more than 80% of the peptide remained in the acidified samples.Addition of ZnCl₂ seems to stabilize PACAP 66 to a higher degree.

FIG. 6 shows the stability of PACAP 66 suspension at 20 mg/mL in2-propanol at 40° C. Addition of ZnCl₂ significantly improved thestorage stability of PACAP 66.

FIG. 7 shows the stability of PACAP 66 in a lyophilized state at 40° C.Acidification significantly stabilized the peptide during storage.Addition of ZnCl₂ seems to stabilize the peptide to a higher degree.

Mechanisms of PACAP 66 Stabilization

(1) Metal Ion-induced PA CAP 66 Stabilization

The results show that ZnCl₂ stabilized PACAP 66 in DMSO, while MgCl₂ andCaCl₂ did not This suggests that metal ions do not stabilize PACAP 66simply by ionic interactions. Therefore, we proposed that zinc and PACAP66 form a chelate complex via the N-terminal histidine residue, whichhinders its own degradation. To prove our hypothesis, we measured theNMR spectrum of PACAP 66 in DMSO in the absence and presence of 1 mMZnCl₂ (FIG. 8). The most dramatic difference in the spectrum in thepresence of 1 mM ZnCl₂ is the disappearance of the histidine H2 and H4signals in the broad amide background. This clearly suggests aninteraction Of ZnCl₂ with the terminal histidine residue. On the otherhand, the spectrum of PACAP 66 in D₂O is essentially the same in theabsence or presence of ZnCl₂ (data not shown). Therefore, these resultsindicate that peptide-Zn interaction is present only in an organicsolvent, not in an aqueous solution, and explain why zinc oxide at 10 mMdid not stabilize PACAP 66 in an aqueous solution (FIG. 1).

The above conclusion on the mechanism of Zn-induced peptidestabilization is supported by data from several references. First, theformation of a metal-peptide complex was observed in PACAP-relatedpeptides. One study showed that several PACAP fragments could form acomplex with copper (II) in an aqueous solution. Kowalik-Jankcowska, etal., J. lnorg. Biochem., 76:63-70 (1999). One of these fragments isHSDGI-NH₂ and the first three amino acids (HSD) corresponds to theN-terminal sequence of PACAP 66. This PACAP fragment forms a dimericcomplex (Cu₂-L₂) between pH 5 to 8 and monomeric complex (Cu-L) above pH8 with a binding ratio of 1:1. It was shown that the third aspartic acidresidue dramatically stabilized the complex. Although these coppercomplexes were identified, it was not mentioned whether the complexwould enhance or compromise the stability of these peptide fragments.Second, zinc is able to form a complex with histidine residues inpeptides, resulting in an altered stability behavior. Zn²⁺ has beenshown specifically to interact with His13 and His14 in amyloidβ-peptide, and the interaction altered the secondary structure of thepeptide and its aggregation behavior. Yang, et al., Eur. J. Biochem.,267:6692-98 (2000). A more recent study showed that binding of Zn²⁺ toamyloid β-peptide(1-16) at a 1:1and 1:2 ratio (peptide/zinc) caused achange (more ordered) in secondary structure, leading to a more stablecomplex. Kozin, et al., supra. Again, the chemical stability of thepeptide could not be predicted. Third, the second residue in PACAP 66 isserine, which has been shown to participate in formation of azinc-peptide complex (Cung, et al., J. Biol. Chem., 263:5574-80 (1988)),and finally, the formation of a zinc-peptide complex may rigidify thepeptide, affecting its stability. Haran, et al., Int. J. Pept. ProteinRes., 20:380-86 (1982).

(2) Acidification-induced PACAP 66 Stabilization

As we discussed before, the major degradation pathway in PACAP 66 inDMSO is dimerization via the formation of a cyclic imide intermediate.It is well known tha the formation of the cyclic imide begins with theintramolecular, nucleophilic attack of the backbone nitrogen on thecarbonyl group of the asparagine side chains. The formation of thecyclic imide is generally accelerated under a basic condition, as abasic condition favors deprotontation of the backbone nitrogen and thedeprotonated nitrogen has a higher nucleophilicity. One the contrary,acidification of the peptide would favor protonation of the backbonenitrogen and slow down the reaction. At the same time, acidificationgenerally facilitates peptide hydrolysis. This was not the case forPACAP 66, however, because the peptide was in a non-aqueous solution,suspension, or dried state.

Implications of the Current Findings

For the first time, we demonstrated that ZnCl₂ can be used as aformulation excipient to stabilize a peptide in an organic solvent, inan organic solvent-based suspension, or in a dried state. Since PACAP66, based on its sequence analysis, is a member of a superfamily ofpeptide hormones, it is anticipated that ZnCl₂ would stabilize anymember of this superfamily in DMSO because of their structuralsimilarities. These member peptides include vasoactive intestinalpeptide (VIP), glucagon, glucagon-like peptides, growth hormonereleasing factor (GRF), secretin, helodermin, and exendin-4. Based uponour analysis of the stabilization mechanisms, ZnCl₂ will also stabilizeany peptide dissolved in DMSO which contains at least one histidineresidue, such as adrenocorticotropic hormone, angiotensins, reninsubstrate tetradecapeptide, natriuretic peptides, gastrointestinalpeptides, luteinizing hormone releasing hormone, melanocyte sitmulatinghormone, and neurotensin, and parathyroid hormone.

Since zinc plays a clear role in the conformational integrity of insulinin the hexameric form and during storage of insulin in an aqueoussolution or suspension, it is probable that zinc will stabilize insulinand other structurally dissimilar polypeptides in an organic solvent, ina solvent mixture, in an organic solvent-based suspension, or in a driedstate.

It has been observed that several PACAP fragments could form a complexwith copper (II) in an aqueous solution. This suggests that othertransition metal ions, in addition to zinc may stabilize PACAP 66 in anorganic solvent, in a solvent mixture, in an organic solvent-basedsuspension, or in a dried state. These transition metal ions mayinclude, but are not limited to, copper, iron, manganese, nickel, andcobalt. Interaction and stabilization by these metals may also beapplicable to other similar or dissimilar peptides, as discussed above.

In this application, we demonstrated stabilization of PACAP 66 atdifferent concentrations in two different organic solvents by metalions. It is very likely that zinc- or other metal-induced stabilizationof PACAP 66, as well as similar or dissimilar peptides, is also operablein other organic solvents or solvent mixtures, including propyleneglycol, s glycerol acetate, monothioglycerol, acetic acid,diethanolamine, benzyl alcohol, ethyl lactate, glycerol formal,N-methylpyrrolidone, polyethyeneglycol 400, isopropyl myristate andother alcohols.

In this application, we also demonstrated stabilization of PACAP 66 byacidification or by combination of acidification and use of metal ionsin an organic solvent, in an organic solvent-based suspension, or in adried state. It is conceivable that PACAP 66 or other peptides(aforementioned) are stabilized by the same strategies in differentorganic solvents (aforementioned), in different solvent mixtures, insuspensions of other organic solvents, and in a dried state. The driedpeptide may be a mixture with any other formulation excipients, deliveryvehicles, or other necessary components. Since acidification stabilizedasparagine residues in PACAP 66, it is conceivable that other peptidescontaining asparagine residues are stabilized by acidification in anorganic solvent, in an organic solvent mixture, in an organicsolvent-based suspension, or in a dried state.

Methods of Use

Formulations of the invention may be used to treat a variety of diseasesand conditions depending on the nature and role of the peptidestabilized. Stabilized formulations of PACAP 66, particularly, maybeused in the treatment of diabetes and related conditions. Formulationsof PACAP 66 may be used alone or in combination with other knowndiabetes treatments. Furthermore, formulations of PACAP 66 may be usedin combination with other therapies to treat diseases or conditionsoften occurring in conjunction with diabetes and related disorders, suchas obesity, lipid disorders and/or hypertension.

The dosage regimen to prevent, treat, give relief from, or ameliorate adiabetic condition or disorder, or to otherwise protect against or treata diabetic condition with the combinations and formulations of thepresent invention is selected in accordance with a variety of factors.These factors include, but are not limited to, the type, age, weight,sex, diet, and medical condition of the subject, the severity of thedisease, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetics and toxicology profilesof the particular inhibitors employed, whether a drug delivery system isutilized, and whether the formulations are administered with otheractive ingredients. Thus, the dosage regimen actually employed may varywidely and therefore deviate from the preferred dosage regimen set forthherein.

The total daily dose of each drug can be administered to the patient ina single dose, or in multiple subdoses. Typically, subdoses can beadministered two to six times per day, preferably two to four times perday, and even more preferably two to three times per day. Doses can bein immediate release form or sustained release form sufficientlyeffective to obtain the desired control over the diabetic condition.

Formulations of the invention containing PACAP 66 may be used to treatdiseases, such as diabetes, including Type 2 diabetes. Such methods mayalso delay the onset of diabetes and diabetic complications. Otherdiseases and conditions that may be treated or prevented usingformulations of the invention include: Maturity-Onset Diabetes of theYoung (MODY) (Herman, et al., Diabetes 43:40 (1994)), Latent AutoimmuneDiabetes Adult (LADA) (Zimmet, et al., Diabetes Med. 11:299 (1994)),impaired glucose tolerance (IGT) (Expert Committee on Classification ofDiabetes Mellitus, Diabetes Care 22 (Supp. 1) S5 (1999)), impairedfasting glucose (IFG) (Charles, et al., Diabetes 40:796 (1991)),gestational diabetes (Metzger, Diabetes, 40:197 (1991), and metabolicsyndrome X.

Formulations of the invention containing PACAP 66 may also be used totreat secondary causes of diabetes (Expert Committee on Classificationof Diabetes Mellitus, Diabetes Care 22 (Supp. 1), S5 (1999)). Suchsecondary causes include glucocorticoid excess, growth hormone excess,pheochromocytoma, and drug-induced diabetes. Drugs that may inducediabetes include, but are not limited to, pyriminil, nicotinic acid,glucocorticoids, phenytoin, thyroid hormone, β-adrenergic agents,α-interferon and drugs used to treat HIV infection.

The formulations of the invention containing PACAP 66 may be used aloneor in combination with additional therapies and/or compounds known tothose skilled in the art in the treatment of diabetes and relateddisorders. Alternatively, the formulations described herein may be used,partially or completely, in combination therapy.

The formulations of the invention containing PACAP 66 may also beadministered in combination with other known therapies for the treatmentof diabetes, including PPAR agonists, sulfonylurea drugs,non-sulfonylurea secretagogues, α-glucosidase inhibitors, insulinsensitizers, insulin secretagogues, hepatic glucose output loweringcompounds, insulin and anti-obesity drugs. Such therapies may beadministered prior to, concurrently with or following administration ofthe formulations of the invention containing PACAP 66. Insulin includesboth long and short acting forms and formulations of insulin. PPARagonist may include agonists of any of the PPAR subunits or combinationsthereof. For example, PPAR agonist may inlcude agonists of PPAR-α,PPAR-γ, PPAR-δ or any combination of two or three of the subunits ofPPAR. PPAR agonists include, for example, rosiglitazone andpioglitazone. Sulfonylurea drugs include, for example, glyburide,glimepiride, chlorpropamide, and glipizide. α-glucosidase inhibitorsthat may be useful in treating diabetes when administered with aformulation of the invention containing PACAP 66 include acarbose,miglitol and voglibose. Insulin sensitizers that may be useful intreating diabetes when administered with the formulations of theinvention containing PACAP 66 include thiazolidinediones andnon-thiazolidinediones. Hepatic glucose output lowering compounds thatmay be useful in treating diabetes when administered with theformulations of the invention containing PACAP 66 include metformin,such as Glucophage and Glucophage XR. Insulin secretagogues that may beuseful in treating diabetes when administered with the formulations ofthe invention containing PACAP 66 include sulfonylurea andnon-sulfonylurea drugs: GLP-1, GIP, PAC/VPAC receptor agonists,secretin, nateglinide, meglitinide, repaglinide, glibenclamide,glimepiride, chlorpropamide, glipizide. GLP-1 includes derivatives ofGLP-1 with longer half-lives than native GLP-1, such as, for example,fatty-acid derivatized GLP-1 and exendin. In one embodiment of theinvention the formulations of the invention containing PACAP 66 are usedin combination with insulin secretagogues to increase the sensitivity ofpancreatic beta cells to the insulin secretagogue.

Formulations of the invention containing PACAP 66 may also be used inmethods of the invention in combination with anti-obesity drugs.Anti-obesity drugs include β-3 agonists, CB-1 antagonists, appetitesuppressants, such as, for example, sibutramine (Meridia), and lipaseinhibitors, such as, for example, orlistat (Xenical).

Formulations of the invention containing PACAP 66 may also be used inmethods of the invention in combination with drugs commonly used totreat lipid disorders in diabetic patients. Such drugs include, but arenot limited to, HMG-CoA reductase inhibitors, nicotinic acid, bile acidsequestrants, and fibric acid derivatives. Formulations of the inventioncontaining PACAP 66 may also be used in combination withanti-hypertensive drugs, such as, for example, β-blockers and ACEinhibitors.

Such co-therapies may be administered in any combination of two or moredrugs (e.g., the formulations of the invention containing PACAP 66 incombination with an insulin sensitizer and an anti-obesity drug). Suchco-therapies may be administered in the form of pharmaceuticalcompositions.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingexamples are included by way of illustration only. Accordingly, thescope of the invention is limited only by the scope of the appendedclaims.

1. A stabilized peptide formulation, either in a solution or in asuspension, comprising: (a) a peptide containing at least one histidineresidue; (b) a transition metal salt; and (c) a pharmaceuticallyacceptable organic solvent.
 2. The formulation of claim 1, wherein saidpeptide is selected from the group consisting of the peptide hormonesuperfamily containing PACAP, PACAP-like peptides, VIP, glucagon,glucagon-like peptides, secretin, helodermin, exendin-4, andfunctionally equivalent variants thereof.
 3. The formulation of claim 1,wherein said peptide is PACAP 66 (SEQ ID NO: 1).
 4. The formulation ofclaim 1, wherein said histidine residue is a terminal histidine residue.5. The formulation of claim 1, wherein said peptide is selected from thegroup consisting of adrenocorticotropic hormone, angiotensins, reninsubstrate tetradecapeptide, natriuretic peptides, gastrointestinalpeptides, luteinizing hormone releasing hormone, melanocyte sitmulatinghormone, and neurotensin, and parathyroid hormone.
 6. The formulation ofclaim 1, wherein said transition metal salt is a salt of a transitionmetal selected from the group consisting of zinc, copper, iron,manganese, nickel and cobalt.
 7. The formulation of claim 6, whereinsaid transition metal salt is a zinc salt.
 8. The formulation of claim1, wherein said organic solvent is selected from the group consisting ofDMSO, 1-methyl-2-pyrrolinidone, propanol, propylene glycol, glycerolacetate, monothioglycerol, acetic acid, diethanolamine, benzyl alcohol,ethyl lactate, glycerol formal, N-methylpyrrolidone, polyethyleneglycol400, and isopropyl myristate.
 9. The formulation of claim 1, whereinsaid organic solvent is a mixture of two or more organic solventsselected from the group consisting of DMSO, 1-methyl-2-pyrrolinidone,propanol, propylene glycol, glycerol acetate, monothioglycerol, aceticacid, diethanolamine, benzyl alcohol, ethyl lactate, glycerol formal,N-methylpyrrolidone, polyethyleneglycol 400, and isopropyl myristate.10. The formulation of claim 8, wherein said organic solvent is DMSO,1-methyl-2-pyrrolinidone, or propanol.
 11. A stabilized peptideformulation, either in a solution or in a suspension, comprising: (a)PACAP 66 (SEQ ID NO: 1) and/or salts thereof; (b) ZnCl₂; and (c) apharmaceutically acceptable organic solvent.
 12. The stabilized peptideformulation of claim 11, wherein said organic solvent is selected fromthe group consisting of DMSO, 1-methyl-2-pyrrolinidone, propanol,propylene glycol, glycerol acetate, monothioglycerol, acetic acid,diethanolamine, benzyl alcohol, ethyl lactate, glycerol formal,N-methylpyrrolidone, polyethyleneglycol 400, and isopropyl myristate.13. The stabilized peptide formulation of claim 12, wherein said organicsolvent is DMSO, 1-methyl-2-pyrrolinidone or propanol.
 14. Theformulation of claim 11, wherein said organic solvent is a mixture oftwo or more organic solvents selected from the group consisting of DMSO,1-methyl-2-pyrrolinidone, propanol, propylene glycol, glycerol acetate,monothioglycerol, acetic acid, diethanolamine, benzyl alcohol, ethyllactate, glycerol formal, N-methylpyrrolidone, polyethyeneglycol 400,and isopropyl myristate.
 15. The formulation of claim 11, wherein saidZnCl₂ is at a ZnCl₂:peptide molar ratio of above 0.1 in said organicsolvent.
 16. The formulation of claim 11, wherein said PACAP 66 and/orsalts thereof are at a concentration of above 0.1 mg/mL of said organicsolvent.
 17. A stabilized peptide formulation, comprising a driedmixture of an acid and a peptide containing at least one asparagineresidue.
 18. The formulation of claim 17, wherein said peptide is PACAP66 (SEQ ID NO: 1).
 19. The formulation of claim 17, wherein said acid isan inorganic acid.
 20. The formulation of claim 19, wherein saidinorganic acid is selected from HCl and H₃PO₄.
 21. The formulation ofclaim 17, wherein said acid is TFA.
 22. The formulation of claim 17,wherein said formulation is freeze-dried or spray-dried.
 23. Theformulation of claim 17, further comprising a transition metal salt. 24.The formulation of claim 23, wherein said transition metal salt is asalt of a transition metal selected from the group consisting of zinc,copper, iron, manganese, nickel and cobalt.
 25. The formulation of claim24, wherein said transition metal is zinc.
 26. A stabilized peptideformulation, comprising a dried mixture of an acid and PACAP 66 (SEQ IDNO: 1) and/or a salt thereof.
 27. The formulation of claim 26, whereinsaid acid is TFA.
 28. The formulation of claim 26, wherein said acid isan inorganic acid.
 29. The formulation of claim 28, wherein saidinorganic acid is selected from HCl and H₃PO₄.
 30. The formulation ofclaim 26, wherein a molar ratio of said acid to said PACAP 66 and/or asalt thereof is above 0.1.
 31. The formulation of claim 26, furthercomprising a transition metal salt.
 32. The formulation of claim 31,wherein said transition metal salt is a salt of a transition metalselected from the group consisting of zinc, copper, iron, manganese,nickel and cobalt.
 33. The formulation of claim 32, wherein saidtransition metal is zinc.
 34. A stabilized peptide formulation,comprising a dried mixture of a transition metal salt and a peptidecontaining at least one histidine residue.
 35. The formulation of claim34, further comprising a pharmaceutically acceptable organic solvent.36. The formulation of claim 35, wherein said organic solvent isselected from the group consisting of DMSO, 1-methyl-2-pyrrolinidone,propanol, propylene glycol, glycerol acetate, monothioglycerol, aceticacid, diethanolamine, benzyl alcohol, ethyl lactate, glycerol formal,N-methylpyrrolidone, polyethyleneglycol 400, and isopropyl myristate.37. The formulation of claim 36, wherein said organic solvent is DMSO,1-methyl-2-pyrrolinidone or propanol.
 38. The formulation of claim 35,wherein said organic solvent is a mixture of two or more organicsolvents selected from the group consisting of DMSO,1-methyl-2-pyrrolinidone, propanol, propylene glycol, glycerol acetate,monothioglycerol, acetic acid, diethanolamine, benzyl alcohol, ethyllactate, glycerol formal, N-methylpyrrolidone, polyethyleneglycol 400,and isopropyl myristate.
 39. The formulation of claim 34, wherein saidpeptide is selected from the consisting of the peptide hormonesuperfamily containing PACAP, PACAP-like peptides, VIP, glucagon,glucagon-like peptides, secretin, helodermin, exendin-4, andfunctionally equivalent variants thereof.
 40. The formulation of claim34, wherein said peptide is PACAP 66 (SEQ ID NO: 1).
 41. The formulationof claim 34, wherein said peptide is selected from the group consistingof adrenocorticotropic hormone, angiotensins, renin substratetetradecapeptide, natriuretic peptides, gastrointestinal peptides,luteinizing hormone releasing hormone, melanocyte sitmulating hormone,and neurotensin, and parathyroid hormone.
 42. The formulation of claim34, wherein said transition metal salt is a salt of a transition metalselected from the group consisting of zinc, copper, iron, manganese,nickel and cobalt.
 43. The formulation of claim 42, wherein saidtransition metal salt is a zinc salt.
 44. A process for preparing astabilized peptide formulation, comprising the steps of: (a) preparingan acid solution of acid and water; (b) cooling said acid solution tobelow room temperature; (c) mixing said cooled acid solution and apeptide containing at least one asparagine residue to create a cooledmixture; and (d) drying said cooled mixture.
 45. The process of claim44, wherein said acid is an inorganic acid.
 46. The process of claim 45,wherein said inorganic acid is selected from HCl and H₃PO₄.
 47. Theprocess of claim 44, wherein said acid is TFA.
 48. The process of claim44, wherein said peptide is PACAP 66 (SEQ ID NO: 1) and/or a saltthereof.
 49. The process of claim 48, wherein a molar ratio of said acidto said PACAP 66 and/or a salt thereof is above 0.1
 50. The process ofclaim 44, wherein said drying step is freeze-drying or spray-drying. 51.The process of claim 44, further comprising adding a transition metalsalt to said cooled mixture before drying said cooled mixture.
 52. Theprocess of claim 51, wherein said transition metal salt is a salt of atransition metal selected from the group consisting of zinc, copper,iron, manganese, nickel and cobalt.
 53. The process of claim 52, whereinsaid transition metal is zinc.
 54. A process for preparing a stabilizedpeptide formulation, comprising the steps of: (a) mixing an aqueoussolution containing a transition metal salt with a peptide containing atleast one histidine residue; and (b) drying said mixture.
 55. Theprocess of claim 54, wherein said peptide is selected from the groupconsisting of the peptide hormone superfamily containing PACAP,PACAP-like peptides, VIP, glucagon, glucagon-like peptides, GRF,secretin, helodermin, exendin-4, and functionally equivalent variantsthereof.
 56. The process of claim 54, wherein said peptide is PACAP 66(SEQ ID NO: 1).
 57. The process of claim 54, wherein said peptide isselected from the group consisting of adrenocorticotropic hormone,angiotensins, renin substrate tetradecapeptide, natriuretic peptides,gastrointestinal peptides, luteinizing hormone releasing hormone,melanocyte sitmulating hormone, and neurotensin, and parathyroidhormone.
 58. The process of claim 54, wherein said transition metal saltis a salt of a transition metal selected from the group consisting ofzinc, copper, iron, manganese, nickel and cobalt.
 59. The process ofclaim 58, wherein said transition metal salt is a zinc salt.
 60. Theprocess of claim 54, further comprising the step of adding apharmaceutically acceptable organic solvent to said dried mixture. 61.The process of claim 60, wherein said organic solvent is selected fromthe group consisting of DMSO, 1-methyl-2-pyrrolinidone, propanol,propylene glycol, glycerol acetate, monothioglycerol, acetic acid,diethanolamine, benzyl alcohol, ethyl lactate, glycerol formal,N-methylpyrrolidone, polyethyeneglycol 400, and isopropyl myristate. 62.The process of claim 60, wherein said organic solvent is a mixture oftwo or more organic solvents selected from the group consisting of DMSO,1-methyl-2-pyrrolinidone, propanol, propylene glycol, glycerol acetate,monothioglycerol, acetic acid, diethanolamine, benzyl alcohol, ethyllactate, glycerol formal, N-methylpyrrolidone, polyethyeneglycol 400,and isopropyl myristate.
 63. The process of claim 61, wherein saidorganic solvent is DMSO, 1-methyl-2-pyrrolinidone, or propanol.
 64. Theprocess of claim 54, wherein said histidine residue is a terminalhistidine residue.
 65. The process of claim 54, wherein said drying stepis freeze-drying or spray-drying.